Apo2 Ligand/TNF-related Apoptosis-inducing Ligand and ...

[CANCER RESEARCH 60, 5754 –5760, October 15, 2000]

Apo2 Ligand/TNF-related Apoptosis-inducing Ligand and Death Receptor 5
Mediate the Apoptotic Signaling Induced by Ionizing Radiation in
Leukemic Cells1

Bendi Gong and Alex Almasan2

Department of Cancer Biology, Lerner Research Institute, and Department of Radiation Oncology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

ABSTRACT (also called TRAIL-R1; Ref. 2), and DR5 (also called Apo2/TRAIL-
R2/TRICK2/KILLER; Refs. 3– 6) molecules (7). Engagement of these
Ionizing radiation is a major tool for cancer treatment. The response of receptors by the FasL (also Apo1L/CD95L), or the apoptosis-inducing
eukaryotic cells to ionizing radiation includes apoptosis, a process which ligand Apo2L (also TRAIL; Refs. 8 and 9) can lead to cell death.
requires activation of multiple genes. We sought to determine whether Expression of Apo2L at mRNA (8) and protein levels (10) was
radiation-induced gene expression plays a role in radiation-induced detected in many human tissues, particularly in those of hematopoietic
apoptosis. We found Apo2 ligand (Apo2L, also called TRAIL) mRNA origin. The receptors for Apo2L, DR4, and DR5 contain a cytoplasmic
induction following ␥-irradiation of Jurkat, MOLT-4, CEM, and PBMC, “death domain” capable of engaging the cell suicide apparatus
all human T lineage-derived cells. Increased Apo2L protein levels were through an adaptor molecule intermediate such as a Fas-associated
found in MOLT-4 and Jurkat cells. Radiation also activated the Apo2L death domain protein; (11). Most importantly, it was reported that
death receptor (DR)5 (also called Apo2, TRAIL-R2, or KILLER) in whereas many human tumor cell lines were sensitive to cell-surface or
MOLT-4 cells, which harbor a wild-type p53. We isolated 1152 bp of 5؅ soluble Apo2L, normal cells were not. This apparent protection of
flanking region of the Apo2L gene and a shorter fragment of 716 bp, both normal cells from the cytotoxic effect of Apo2L is believed to be
of which showed promoter activity driving the expression of a luciferase based on a unique set of DcRs; these cells either lack the DcR1 (also
reporter gene; however, the response to radiation in MOLT-4 cells was TRAIL-R3/LIT; Refs. 3–5, and 12) or have a truncated DcR2 (also
lost when only 430 bp of 5؅ proximal flanking sequence was maintained. TRAIL-R4/TRUNDD; Refs. 13 and 14) so they are unable to signal,
Exogenous Apo2L induced phosphatidylserine exposure on cell mem- but compete instead for receptor-binding to Apo2L. An alternative
branes, caspase 8 and caspase 3 activation, key markers of apoptosis, view is that levels of an intracellular inhibitor of caspases, a FLICE-
confirming that the Apo2L/DR5 pathway is functional in these cells. Bid, inhibitory protein (15), may provide resistance in normal cells (16).
a Bcl-2 family protein also known to contribute to receptor-mediated The activation of this receptor-mediated cell death pathway during
apoptosis, was also activated. To determine whether Apo2L and DR5 were cancer therapy is still not well understood.
critical for radiation signaling to apoptosis, we stably expressed a domi-
nant negative DR5⌬-receptor in Jurkat cells. Cell survival was signifi- The present experiments were designed to examine the role of
cantly augmented, indicating that increased Apo2L expression contrib- Apo2L and DR5 in ␥-irradiation-triggered apoptosis. We present
uted to radiation-induced apoptosis. Clonogenic assays demonstrated that evidence that radiation induces both Apo2L and DR5, with Apo2L
purified, recombinant soluble Apo2L enhanced the lethality of low, ther- induction being detected only in T lineage-derived cells. Apo2L
apeutic doses (1–2 Gy) of ␥-irradiation. These data suggest that produc- regulation is mediated through 5Ј flanking sequences we have isolated
tion of Apo2L may cooperate synergistically with the cytotoxic effect of and characterized. Purified Apo2L induces caspase-dependent apop-
radiation, and that combinations of Apo2L and radiation may become a tosis, and when combined with low doses of radiation, it significantly
powerful tool in clinical therapy. enhances cell death, indicating that combinations of Apo2L and
therapeutic radiation may be useful in clinical cancer therapy.
INTRODUCTION

The response of eukaryotic cells to ionizing radiation includes cell

cycle arrest and apoptosis, processes which require activation of MATERIALS AND METHODS
multiple genes. We have previously reported that expression of Bax,
a death agonist activated by the p53 tumor suppressor protein, was an Cell Culture and Treatments. Human acute T lymphoblastic leukemia
important effector of radiation-mediated cell death of leukemic cells MOLT-4 and CEM-CM3, and the T-cell leukemia Jurkat were grown in RPMI
(1). However, inhibitors capable of preventing radiation-triggered Bax 1640. A549 lung carcinoma cells were grown in DMEM. All media contained
up-regulation were not able to completely block cell death, suggesting 10% (vol/vol) heat-inactivated FCS, 50 units/ml penicillin, and 50 mg/ml
that factors other than Bax were also involved in radiation-induced streptomycin (Life Technologies, Inc., Gaithersburg, MD). All cells were
cell death. obtained from the American Type Culture Collection (Rockville, MD). Expo-
nentially growing cells were adjusted to a density of 1–2 ϫ 105 cells/ml the day
An alternative pathway to apoptosis could be mediated by surface before the experiment was performed. Human PBMCs were isolated by cen-
receptor signaling, which depends on ligand-receptor interactions for trifugation over Ficoll-Hypaque density gradient sedimentation (Amersham-
cell death effector activity. These cell surface receptors are part of the Pharmacia Biotech, Piscataway, NJ) from blood samples obtained from
TNF3-receptor family and include Fas (also called CD95/Apo1), DR4 healthy adult donors. Jurkat cells were transfected with pcDNA3-DR5⌬ (res-
idues 1 to 268), as described (17). DR5⌬ lacks the death domain, and has been
Received 12/10/99; accepted 8/9/00. shown to function as a dominant-negative inactivating the function of the
The costs of publication of this article were defrayed in part by the payment of page endogenous DR5 (4). DR5⌬ also contains a FLAG epitope-tag which facili-
charges. This article must therefore be hereby marked advertisement in accordance with tates examination of its expression levels. Transfected cells were selected in
18 U.S.C. Section 1734 solely to indicate this fact. the presence of 1 mg/ml G418 (Life Technologies, Inc.)-containing medium
1 This work was supported by research grants from NIH (CA81504 and CA82858). and subsequently maintained with 0.5 mg/ml of G418.
2 To whom requests for reprints should be addressed, at Department of Cancer
Biology, NB40, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH Irradiation was performed as described previously (1) with a 137Cs source
44195. Phone: (216) 444-9970; Fax: (216) 445-6269; E-mail: [email protected]. emitting at a fixed-dose rate of 2.8 Gy/min. The Fas response was examined
3 The abbreviations used are: TNF, tumor necrosis factor; Apo2L, Apo2 ligand; ApoP,
Apo2L promoter; DR, death receptor; Fas, CD95/Fas/Apo1; FasL, Fas ligand; DcR, decoy acetyl-Ile-Glu-Thr-Asp-pNA; DEVD, acetyl-Asp-Glu-Val-Asp-pNA; Gy, gray; FACS,
receptor; PBMC, peripheral blood mononuclear cell; GAPDH, glyceraldehyde-3-phos- fluorescence-activated cell sorter; TRAIL, TNF-related apoptosis-inducing ligand.
phate dehydrogenase; RT-PCR, reverse transcription-PCR; pNA, p-nitroanilide; IETD,

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RECEPTOR-MEDIATED CELL DEATH IN IRRADIATED TUMOR CELLS

using the Fas agonistic mAb (clone CH11; Panvera, Madison, WI) and Fas-Fc ␮g/ml lysozyme. The supernatant was collected after centrifugation at
with enhancer (Alexis Corporation, San Diego, CA). All chemicals, unless 14,000 ϫ g for 15 min and used for additional purification. The recombinant
specified, were obtained from Sigma Chemical Co. (St. Louis, MO). soluble Apo2L (used in Fig. 6) was then affinity-purified by His6-resin affinity
purification following the manufacturer’s instructions (Novagen, Milwaukee,
RNA Analyses. Total RNA was isolated from cells at various intervals WI). Apo2L protein specificity was confirmed by SDS-PAGE and Western
post-irradiation using the Trizol reagent (Life Technologies, Inc.). To blot analysis with an anti-Apo2L antibody (C19, Santa Cruz Biotechnology,
determine the steady-state levels of RNA, we used the RiboQuant system Santa Cruz, CA). For some experiments (Figs. 4 and 5) a commercial
(PharMingen, San Diego, CA) for RNase protection assay with multiprobe rhsApo2L was used, containing the extracellular domain of Apo2L fused at the
template sets which allow simultaneous quantitation and characterization of NH2 terminus to a FLAG-tag and an 8-amino acid linker peptide. A combi-
multiple RNA molecules, as described (1). The hAPO-3 and hAPO-3C tem- nation of Apo2L protein and enhancer have been used according to manufac-
plate sets (PharMingen) were used for the T7 polymerase-directed synthesis of turer’s specifications (Alexis Corporation, San Diego, CA).
high specific activity [32P]-labeled antisense RNA probes. The hAPO-3 probe
set contains the following genes: (a) the receptors DR3 and TNFRp55; (b) the Immunoblotting and Antibodies. Cell lysates were resolved by one-
ligands FasL and Apo2L; (c) the adapters FLICE, Fas-associated death do- dimensional SDS-PAGE under reducing conditions, as described (1). The blots
main; and RIP (receptor-interacting protein, which interacts with Fas), as well were then incubated with primary antibodies from PharMingen (Apo2L; 2
as FAF (Fas-associated protein) and FAP (PNP1 protein tyrosine phosphatase ␮g/ml), Sigma (FLAG M5, 1:1000 dilution; ␤-actin, 1:5000 dilution), Santa
1E). hAPO-3C contains the DR4, DR5, and DcR1 receptors for Apo2L. Both Cruz (caspase 3 p20; 1:500 dilution; caspase-8 H277; 1:500 dilution), or from
sets contain the housekeeping gene L32 as an internal control. X. Wang [University of Texas Southwestern (20); Bid polyclonal, 1:1000
dilution) in Tris-buffered saline with 0.05% Tween 20 containing 5% nonfat
To amplify DcR2, the sense 5Ј-catgggactttggggacaaa-3Ј and antisense 5Ј- dry milk for 16 h at 4°C. The blots were then washed in Tris-buffered saline
gatagggagaggcaagcatc-3Ј primers were used as well as those for GAPDH with with 0.05% Tween 20, followed by incubation with the secondary antibody
RNA isolated as above. The RT-PCR reaction was carried out as described (1). conjugated to horseradish peroxidase, and developed using the enhanced
chemiluminescence detection system (Amersham, Arlington Heights, IL) fol-
Promoter Activity. By searching the National Center for Biotechnology lowed by exposure to X-ray film (Eastman Kodak).
Information using the Basic Local Alignment Search Tool, we found that the
Homo sapiens chromosome 3, clone hRPK.44_A_1 (accession no. RESULTS
AC007051), contained the whole Apo2L/TRAIL gene (five-exon structure) as
well as the 5Ј upstream promoter region. The primers for the ApoP, ApoP/ Ionizing Radiation Induces Apo2L in T Lineage-derived Cells.
1152, 5Ј-gcctcgaggaatctgggaggtggagg-3Ј, ApoP/716, 5Ј-gcctcgagctgtccagc- We sought to examine whether radiation affected expression of cell
ctaacacac-3Ј, ApoP/430, 5Ј-gcctcgaggtgcctatgacagccagg-3Ј, and ApoP/anti- DRs, their specific ligands and interacting partners, known to be
sense, 5Ј-cgaagcttgatcctgtcagag tctgac-3Ј were used to generate, by PCR, the involved in cell-surface receptor-mediated apoptosis (7). The mRNA
fragments of the ApoP. The PCR reaction was done using the Expand High expression of PBMC and Jurkat, representing normal and tumor T
Fidelity PCR System (Roche Molecular Biochemicals, Indianapolis, IN) fol- cell-derived cells, were examined using a multiprobe RNase protec-
lowing the manufacturer’s protocol. MOLT-4 genomic DNA, isolated by tion assay. Jurkat cells were chosen because they do not have func-
QIAamp Tissue Kit (QIAGEN, Valencia, CA), was used as the template. The tional p53 and, therefore, radiation does not activate p53-target genes
amplified 1152-, 716-, and 430-bp DNA fragments were subcloned into the known to be induced during apoptosis, such as Bax. Most signifi-
pGL2 plasmid containing the luciferase reporter gene (Promega, Madison, cantly, we found a 1.8-fold up-regulation of Apo2L at 4 h following
WI), and their nucleotide sequence was confirmed by DNA sequence analysis, 2 Gy of ␥-irradiation in PBMCs. Moreover, Apo2L expression was
as described (1). also induced two-fold at 4 h post-irradiation in Jurkat cells (Fig. 1).
Similar results were obtained with another T cell-derived tumor cell
The human ApoP activity was determined in MOLT-4 cells, as described line, CEM-CM3, but not in several other human tumor cell lines (data
(17), after cotransfection with 1 ␮g of Apo2L luciferase reporter and 0.1 ␮g of not shown). These data indicate that radiation-induced Apo2L expres-
pCMV-LacZ plasmid DNA. At 32 h after transfection with the DMRIE-C sion was observed in both normal as well as T cell-derived tumor cell
reagent (Life Technologies, Inc.), the cells were irradiated, and 4 h later, lines.
luciferase activity was determined and normalized to values of ␤-gal. Each
assay was performed in triplicate. In addition to Apo2L, we found that Fas levels were also up-
regulated in PBMCs, whereas FasL expression remained unchanged.
Apoptosis, Cytotoxicity, and Clonogenic Assays. Phosphatidylserine ex- Interestingly, PMBC was the only cell type in which FasL levels were
posure on cell membranes was determined with FITC-Annexin V by flow high enough to be detected by RNase protection. As expected, Jurkat
cytometry as described (1, 18) using a FACScan and analyzed with CellQuest cells, which lack functional p53, did not up-regulate Fas following
software (Becton Dickinson, San Jose, CA) on mean values obtained from the irradiation, confirming the reported p53 requirement for Fas up-
cell population from which debris were gated out. Caspase activity was regulation (21). Taken together, these results indicate that radiation-
measured as described (1), using the IETD- and DEVD-pNA-derived chromo- induced Apo2L expression was T-lineage dependent, but independent
genic substrates for caspase 8 and -3 activity, by enzyme-catalyzed release of of wild-type p53 function.
pNA monitored at 405 nm.
Activation of the Apo2L/DR5 Pathway by Ionizing Radiation in
␥-irradiation and recombinant soluble Apo2L-treated cells were seeded MOLT-4 Cells. To further characterize Apo2L regulation, we next
(2–3 ϫ 104 cells) in 96-well plates. Cell viability was examined 24 h later with examined its expression in MOLT-4 cells, which we have previously
the CellTiter 96 Aqueous One Solution Reagent (MTS; Promega) to measure characterized for the role of p53-dependent bax induction in apoptosis
tetrazolium reduction, as indicated by absorbance at 490 nm using an ELISA (1). Control and irradiated MOLT-4 cells were examined at 2 to 8 h
reader (Spectramax 340). All determinations were done in triplicate. after ␥-irradiation, a time period during which no cytotoxic effects
were observed. Most significantly, we found that Apo2L mRNA was
For clonogenic assays, recombinant soluble Apo2L was added 16 h before induced significantly after 10 Gy of ␥-irradiation (Fig. 2A, top). There
␥-irradiation to U-bottomed 96-well plates with each well containing 4 cells/ was no detectable Apo2L expression in untreated MOLT-4 cells or in
100 ␮l of medium. Cell colonies were counted 14 days later. Cellular clono- those examined 8 h post-irradiation, indicating a transient radiation-
genicity was calculated by the formula: % Clonogenicity ϭ (ln ϫ [96/Neg dependent regulation. Moreover, there was no significant change in
Wells]) (plate density) ϫ 100, where Neg Wells represent the number of wells the expression levels of most of the other transcripts, including the
that have failed to grow to 50 cells (19). MOLT-4 and Jurkat cells have a housekeeping gene L32, indicating a specific change in expression
cloning efficiency of 40% and 66.5%, respectively. Ps were determined by the
t test using Microsoft Excel.

Preparation of Recombinant Soluble Apo2L/TRAIL. Escherichia coli
BL21 strain was transformed with a His6-tagged pET28b-TRAIL (residues 95
to 281), a generous gift from Drs. E. S. Alnemri and S. M. Srinivasula (4).
Expression of the protein was induced with 0.4 mM isopropyl-1-thio-␤-D-
galactopyranoside for 2 h, and bacteria were pelleted and sonicated in a buffer
containing 50 mM Tris-HCl, (pH 8.0), 2 mM EDTA, 0.1% Triton X, and 100

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consistent with that detected for RNA levels. This increase was
specific to Apo2L, as there were no changes in the levels of ␤-actin

(Fig. 2D).

To determine the effect of promoter sequences on Apo2L regula-
tion, we isolated 1152 bp of 5Ј-flanking Apo2L DNA sequence

upstream of the Apo2L translation initiation site and created two
deletion promoter constructs: ApoP/716 and ApoP/430. Each con-
struct was used to transfect MOLT-4 cells, which were then either left
untreated or subjected to 10 Gy of ␥-irradiation. Compared with

control, the expression of the luciferase gene driven by ApoP/1152

was induced up to 2.3-fold by irradiation (Fig. 3). Similarly, ApoP/

716 showed a substantial, up to 1.8-fold, increase in promoter activity
in irradiated cells. In contrast, there was no significant change in the
expression of the luciferase gene driven by ApoP/430 under similar
conditions. These data suggest that the 5Ј-flanking Apo2L DNA
sequence contained between nucleotides Ϫ1152 and Ϫ430 is required

for radiation-induced ApoP activity.

Radiation-induced Apoptosis Is Dependent on Apo2L/DR5

Function. Apo2L binds to several receptors, of which only DR5 is
expressed in Jurkat and MOLT-4 cells (Fig. 2). To examine the extent
to which Apo2L expression contributes to cell death, we stably
expressed a FLAG-tagged dominant-negative DR5⌬ into Jurkat cells;

these cells were chosen because no other known death effectors are

induced. In contrast, multiple death effectors are up-regulated follow-

ing irradiation of MOLT-4 cells, such as Bax (1), and therefore it
would be difficult to dissociate the effect of Apo2L from that of other
death-promoting proteins.

Fig. 1. Ionizing radiation induces Apo2L mRNA in T lineage-derived cells. The If Apo2L expression had a role in apoptosis, then blocking the
steady-state mRNA expression in PBMC and Jurkat cells was examined in either un- Apo2L signaling pathway should have an impact on cell survival.
treated cells or in those 4 h after irradiation (2 Gy for PMBC and 20 Gy for Jurkat) by Radiation treatment induces phospatydylserine exposure on the cell
multiprobe RNase protection assay with the hAPO-3 probe set, as described in “Materials membrane, a relatively early marker of apoptosis (1, 18). We found
and Methods.” The mRNA levels are shown, with the fold induction indicated at the that 27% of Jurkat cells were viable, as they did not stain with
bottom, and the size of the protected RNA products indicated on the right. All data are Annexin V (which binds to phospatydylserine), 24 h after 20 Gy of
from a single gel, representing values obtained by normalizing levels of mRNA obtained ␥-irradiation. In contrast, 56% of Jurkat/DR5-⌬ cells survived (Fig.
after irradiation to those of untreated cells and L32, using the ImageQuant program.

4A). Moreover, there was a marked difference in survival between
levels of Apo2L. A time and dose-response experiment indicated a parental and DR5⌬-expressing Jurkat cells as early as 12 h following
transient increase in Apo2L expression at all radiation doses tested irradiation (Fig. 4B). Thus 67, or 54%, of Jurkat/DR5⌬ cells survived,

(2–10 Gy; Fig. 2B). However, the magnitude of this response, a 2.2- compared with 50, or 31%, of irradiated parental cells examined at 12
to 3-fold increase at the lower 2– 4 Gy dose, was considerably less or 24 h, respectively. These cells expressed the DR5⌬ receptor, as
than the 8.5-fold increase obtained after 10 Gy. Similarly, Fas was shown by the presence of the FLAG epitope with which they were

induced up to eight-fold at doses as low as 2 Gy (at 8 h following tagged (inset). Fas-Fc pretreatment had no significant effect on sur-
irradiation) compared with untreated cells. Treatment with 4 or 10 Gy vival of DR5⌬/Jurkat cells and only a slight effect on survival of
␥-irradiation resulted in higher induction levels with a more substan- Jurkat cells 12 h after treatment. Furthermore, the Jurkat/DR5⌬ cells

tial increase at earlier (4.5- versus 2.5-fold at 2 h) compared with later were resistant to the cytotoxic effect of Apo2L, but remained sensitive
times (8 to 10.5-fold at 8 h; Fig. 2B). These results indicate a time and to killing by an anti-Fas agonisitc antibody, indicating the specificity
dose-dependent induction of both Apo2L and Fas, but with a different of DR5⌬ function (Fig. 4C). Fas-Fc pretreatment had no significant
kinetics of induction of the two genes. effect on survival of Apo2L-treated DR5⌬ cells, but prevented, as
expected, the cell death of Fas-treated cells. These results indicate that
We next sought to examine the expression and regulation of the

receptors that mediate Apo2L signaling. We found that only DR5 was blocking the DR5-mediated Apo2L signaling has a significant impact
expressed in MOLT-4 and Jurkat cells (Fig. 2A, bottom), with no on cell survival and that ␥-irradiation-induced Apo2L expression

detectable DR4 and DcR1 present (data not shown). Following irra- contributes to the cell death of hematopoietic cells.
diation, DR5 levels were increased in MOLT-4 cells, which harbor Apo2L Induces Apoptosis through Activation of Caspase 8, Bid,
wild-type p53, but not in Jurkat cells, which have a p53 mutation. and Caspase 3. Apo2L can induce apoptosis in a variety of tumor

Because Apo2L was reported to also signal through DcR2, we exam- cells. However, the mechanism of Apo2L-induced apoptosis remains
ined its expression by RT-PCR. There was no DcR2 expression in to be clarified. Apo2L induced rapid apoptosis of Jurkat cells in a

either MOLT-4 or Jurkat cells, either before or after radiation treat- time-dependent manner. Cells started to loose viability as early as 4 h

ment. In contrast, under the same experimental conditions, DcR2 was after treatment, with most of cells being Annexin V-FITC-positive by
readily detectable in A549 lung carcinoma cells (Fig. 2C), as reported 12 h (Fig. 5A). To assess whether caspases were activated in our
previously (13), whereas the levels of GAPDH, an internal control, system, we determined caspase activity in Apo2L-treated cells. Apo2L
were unchanged. induced effectively IETD- and DEVD-pNA cleavage activity, corre-

Immunoblot analyses, using anti-Apo2L antibodies, showed that sponding to caspase 8 and caspase 3; this activity started at 2 h and

Apo2L protein levels were also up-regulated 2- to 3.5-fold in irradi- reached a maximum at 4 – 6 h (Fig. 5B). Moreover, Western blot

ated Jurkat and MOLT-4 cells, respectively, a pattern of induction analyses show that the procaspase 3 was proteolytically cleaved to the

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Fig. 2. Regulation of the Apo2L/DR5 pathway
by ionizing radiation. A, MOLT-4 cells were either
untreated or irradiated (10 Gy) and the steady-state
mRNA expression was analyzed similarly to Fig. 1
(top). DR5 mRNA expression was analyzed simi-
larly, except that the hAPO-3C probe set was used
(bottom). B, the dose and time-dependent expres-
sion of Apo2L and Fas were determined by Phos-
phorImager analyses. C, total RNA was isolated, as
above, before and 4 h after irradiation and sub-
jected to RT-PCR analysis for DcR2 and GAPDH,
as described (1). D, immunoblot analyses were
done as described in “Materials and Methods” on
cells lysed at the indicated times after irradiation of
MOLT-4 and Jurkat cells, using anti-Apo2L and
anti-␤-actin antibodies. The Apo2L protein levels
were quantitated using NIH Image following nor-
malization to ␤-actin.

p17 kDa fragment, known to be the activated form of caspase 3. To together with the increase in IETD-pNA cleavage activity, indicate
address the pathway by which caspase 3 was activated, we examined that caspase 8 is activated by Apo2L. Finally we examined Bid, a
in more detail caspase 8, an apical caspase known to be the first Bcl-2 family member also recently reported to be involved in recep-
caspase to be activated in receptor-mediated apoptosis. Levels of tor-mediated apoptosis and to further caspase 3 activation (20, 22).
procaspase 8 were decreased substantially by 4 h, with further time- Bid levels were significantly reduced upon Apo2L treatment, indicat-
dependent decrease in protein levels (Fig. 5C). These results, taken ing processing of the full-length Bid to a p15- or p13-kDa fragment

not detectable under our experimental conditions. Activation of

caspase 8 and caspase 3 indicates a caspase-dependent apoptosis, with
Bid most likely contributing to the amplification of the caspase

cascade.

Combination of Apo2L and Radiation Treatments Greatly En-

hance Lethality. To further examine a direct contribution of Apo2L

induction to cell death, we affinity-purified recombinant, soluble

Fig. 3. Apo2L promoter activity following irradiation. MOLT-4 cells were cotrans- Apo2L protein, which was shown by SDS-PAGE to be present as a
fected with the ApoP/1152, ApoP/716, or ApoP/430 promoter proximal fragments and 28-kDa monomeric protein (Fig. 6A). Apo2L protein specificity was
cloned into the pGL2 plasmid and pCMV-LacZ. Thirty-two h after transfection, cells were confirmed by Western blotting with an anti-Apo2L antibody. Using a
irradiated (10 Gy), and the luciferase activity was measured 4 h later. The assays were concentration of 2–5 ␮g/ml of Apo2L, we found that both MOLT-4
normalized to the values obtained for ␤-Gal activity. Each assay was performed in and Jurkat cells could be effectively killed in a dose-dependent
triplicate. manner (Fig. 6B).

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Fig. 4. DR5⌬ blocks radiation and Apo2L-
induced apoptosis. A, expression of phospatydyl-
serine on the plasma membrane was measured by
staining cells with FITC-labeled Annexin V, in
conjunction with PI. At 24 h following 20 Gy of
␥-irradiation, cells were prepared for flow cytom-
etry. The data were obtained from the cell popula-
tion from which debris were gated out against
forward- and side-scatter. These data are represent-
ative of two separate experiments. B, parental or
DR5⌬-expressing Jurkat cells were irradiated, in
the absence or presence of Fas-Fc (1 ␮g/ml), and
the proportion of viable cells, which did not stain
with Annexin V, was determined by FACS 12 or
24 h after 20 Gy of ␥-irradiation. Inset, immunoblot
analyses in Jurkat cells or those expressing DR5⌬-
FLAG using anti-FLAG or anti-␤-actin antibodies.
C, parental or DR5⌬-expressing Jurkat cells, in the
absence or presence of Fas-Fc, were treated with
Apo2L (100 ng/ml) or agonistic Fas antibodies
(CH11; 10 ng/ml). The proportion of viable cells,
was determined by FACS 12 h after treatment.

To further examine the possible clinical effects of Apo2L, clono- Apo2L resulted in a decreased survival from 55.9 Ϯ 8% to
genic assays were next performed on cells treated with radiation or 29.5 Ϯ 2.6% or 7.3 Ϯ 1.7% (P Ͻ 0.01) for the 1- or 2-Gy dose,
Apo2L alone or the two agents used in combination. We choose a 10- respectively. When higher concentrations of Apo2L (0.2 ␮g) were
to 20-times lower concentration of Apo2L protein, which did not have used, survival decreased from 52.2 Ϯ 6.8% to 22.9 Ϯ 4.8% or
any detectable cytotoxic effects. Thus, MOLT-4 and Jurkat cells were 6.7 Ϯ 0.9% (P Ͻ 0.01) at the two radiation doses. These data indicate
treated with 0.1– 0.2 ␮g/ml of Apo2L for 16 h, then irradiated with a a statistically significant (t test) decrease in clonogenic survival of
dose of 1–2 Gy. The radiation survival of MOLT-4 and Jurkat cells,
MOLT-4 and Jurkat cells treated with a combination of Apo2L and

which contain a functional or mutated p53, respectively, is shown in radiation as compared with radiation alone. These results indicate a
Fig. 6. Low concentrations of Apo2L resulted in only a slight reduc- synergistic interaction between Apo2L and radiation in reducing

tion in clonogenic survival of MOLT-4 and Jurkat cells. Clonogenic clonogenic survival of these hematopoietic tumor cell lines.
survival decreased slightly, however, in MOLT-4 cells, from 40 Ϯ 4.3
to 33 Ϯ 3.5% following 1 Gy of ␥-irradiation (Fig. 6C). In contrast, DISCUSSION
combined treatment with radiation and Apo2L reduced the clonogenic
survival from 34.5 Ϯ 3.2% to 20 Ϯ 2.2% (P Ͻ 0.5) and from In this study we report that ionizing radiation, an important modal-
35.9 Ϯ 4.8% to 16 Ϯ 2.8% (P Ͻ 0.01), respectively, for the two ity for cancer treatment, induces up-regulation of the gene products
Apo2L concentrations. For Jurkat cells, survival was reduced from for the ligand Apo2L and of its receptor DR5. These are key regula-
66.5 Ϯ 4.7% to 41.3 Ϯ 3.7% or 16.8 Ϯ 1.8% when 1 or 2 Gy of tory proteins that participate in a receptor-mediated cell-death path-
␥-irradiation were used (Fig. 6D). Use of radiation and 0. 1 ␮g/ml of way. This report represents the first observation of Apo2L regulation
by radiation, detected at both the mRNA and protein levels in T

lineage-derived tumor and normal human cells. A dominant negative
DR5⌬ significantly prevented radiation-induced cell death, demon-

strating the important contribution of Apo2L/DR5 expression to
radiation-induced apoptosis. This finding is consistent with a recent
suggestion that radiation kills lymphocytes by a Fas-independent
mechanism (23). Apo2L induction in T cells could be most important
because radiation could selectively kill tumor cells, which express

only the DR5 and/or DR4 receptors, but not normal cells, which also
express DcRs or FLICE-inhibitory protein.

To elucidate the mechanism of Apo2L regulation, we isolated and
characterized the 5Ј-flanking region of the human Apo2L gene, which
showed promoter activity in irradiated MOLT-4 cells. The consensus
sequences for several transcription-regulatory factor-binding motifs
were identified to be present in ApoP/1152, but not the ApoP/430

Fig. 5. Activation of caspases and Bid and induction of apoptosis by recombinant promoter region, including sites for OCT-1, SP-1, GATA1–3, and
Apo2L. A, induction of apoptosis in Jurkat cells was examined by Annexin V staining, as C/EBP. These sites, located in the promoter region between nucleo-
above, at 4, 8, and 12 h following treatment with 100 ng/ml Apo2L. B, the IETD-pNA and tides Ϫ1152 to Ϫ716 (OCT-1, SP-1, GATA1-3), and Ϫ716 to Ϫ430
DEVD-pNA cleavage, as a measure of caspase 8 and caspase 3 activity, was determined (C/EBP) could mediate the radiation-caused transcriptional induction
after 2– 8 h of treatment, as described in “Materials and Methods.” C, Jurkat cells were of Apo2L. In fact, a DNA-binding activity was reported in several
treated with Apo2L for 4 – 8 h and subjected to Western blotting for caspase 8, caspase 3, tumor cell lines following irradiation for both OCT-1 and SP-1 (24).
and Bid, using the corresponding antibodies and ␤-actin as a control. The 17-kDa The exact role of these elements for the radiation response needs to be
fragment recognized by the anticaspase 3 antibody represents the activated form of investigated further.
caspase 3.
A functional role for Fas in the radiation response has been reported
in T cells from animals with defective expression of Fas or FasL

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RECEPTOR-MEDIATED CELL DEATH IN IRRADIATED TUMOR CELLS

Fig. 6. Clonogenic survival of Apo2L and radi-
ation-treated cells. A, recombinant Apo2L was ob-
tained as described in “Materials and Methods.”
Apo2L protein specificity was confirmed by SDS-
PAGE and Western blot analysis with an anti-
Apo2L antibody. B, MOLT-4 and Jurkat cells were
treated with different concentrations of recombi-
nant Apo2L. Induction of cell death was examined
by the MTS cell proliferation assay to determine
the percentage of biochemically active cells after
24 h of treatment. MOLT-4 (C) and Jurkat cells (D)
were placed in U-bottomed 96-well plates with
each well containing 4 cells/well in 100 ␮l me-
dium. Cells were treated with radiation with or
without recombinant, soluble Apo2L (0.1 or 0.2
␮g/ml). Cell colonies were counted 14 days later
and the clonogenic survival calculated as described
in “Materials and Methods.” P Ͻ 0.05 (‫ )ء‬or 0.01
(‫)ءء‬, respectively. Ps were determined by t test
using Microsoft Excel. n ϭ 6; bars, SE.

function, which showed reduced sensitivity to radiation-induced ap- The observations that radiation can induce Apo2L and that low

optosis (25). Fas was induced in our system too, consistent with its doses of radiation can cooperate synergistically with Apo2L in en-

up-regulation, in a p53-dependent manner, following treatment with hancing cell death may have implications for clinical therapy. This is

DNA-damaging and oxidant agents of a number of hematopoietic and reminiscent of the synergistic or additive cell killing between TNF

solid tumor cell lines (21). However, MOLT-4 cells were resistant to and radiation reported in several solid tumor cell lines (31). Gene

Fas treatment (500 ng/ml for 24 h, data not shown). Moreover, in therapy approaches have been proposed based on radiation-responsive

Jurkat cells, which harbor mutant p53, Fas was not induced by promoters driving TNF expression (32), and TNF expression has been

radiation, there was no detectable FasL mRNA present, and Fas-Fc shown to sensitize certain radiation-resistant tumors (33). In addition,

pretreatment had no effect on radiation-induced cell death. Most there are essential differences between the Apo2L-DR4 or DR5 path-
importantly, DR5⌬ protected Jurkat cells from radiation and Apo2L- way and those using TNF and FasL, which further indicate that

induced, but not Fas-induced, cell death, indicating that the Fas/FasL Apo2L might be a safer agent. Thus, at least in some systems, it has

system is not involved in radiation-induced apoptosis in our system, been shown that Apo2L kills tumor cells and not normal cells because

although it is clearly important for regulation of apoptosis in some of the presence on the latter of DcRs (3, 5). More recently, the

other biological systems. This finding is consistent with reports that observations of a lack of cytotoxicity of Apo2L toward normal cells

radiation kills lymphocytes by a Fas-independent mechanism (23) and and tissues have been extended successfully to mice (34) and nonhu-

that chemotherapy-induced apoptosis is not dependent on Fas/FasL man primates (35). Several chemotherapeutioc agents have been also

interactions (26, 27). In fact, various human lymphoid cell lines differ shown to have a synergistic activity with Apo2L (35, 36), indicating

in their sensitivity toward induction of apoptosis by Apo2L and Fas that combination therapies using Apo2L are likely to be widely

(28), an observation we have extended to myeloid cells.4 applicable.

Radiation induces caspase 8 and -3 activation in Jurkat (data not The present investigations enhance our understanding of the mech-

shown) and MOLT-4 cells, leading to proteolytic cleavage of cellular anism of radiation and Apo2L-induced apoptosis. The observed syn-

proteins such as poly(ADP-ribose)polymerase (1). Treatment of Jur- ergy between Apo2L and low, therapeutic doses of radiation can form

kat cells with purified Apo2L also induced caspase 8 and -3 cleavage the basis for developing strategies for pharmacological intervention,

and activation, similar to previous reports (28, 29), indicating that with potential for clinical application. In particular, the specificity of

radiation may mediate its apoptotic effects by engaging the caspase Apo2L cytotoxicity for tumor cells and its systemic distribution

cascade not only through increased levels of the DR5 receptor, but reaching metastases and the surgical precision with which ionizing

also by increasing the levels of Apo2L. Bid activation indicates the radiation can now be delivered in the clinic could constitute a very

requirement for caspase amplification in the Apo2L/DR5 apoptotic attractive combination for enhanced clinical response of tumors re-

pathway by a mechanism reported to take place through translocation sistant to radiation therapy.

of activated Bid to mitochondria, facilitating cytochrome c release and

activation of caspase 9 and then caspase 3 (20, 22, 30).

ACKNOWLEDGMENTS

4 Q. Chen, B. Gong, A. Zhou, E. Hsi, E. Hsi, M. Hussein, and A. Almasan. Induction We thank Drs. E. S. Alnemri and S. M. Srinivasula for the pET28b-TRAIL
of Apo2L and modulation of Bcl-2-related proteins regulate type I interferon-induced and pCDNA3-DR5⌬ constructs, X. Wang (U. T. Southwestern) for the Bid
apoptosis in multiple myeloma, submitted for publication.

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RECEPTOR-MEDIATED CELL DEATH IN IRRADIATED TUMOR CELLS

antibody, and Amy Raber (Cleveland Clinic Flow Cytometry Core) for expert 17. Gong, B., and Almasan, A. Differential upregulation of TP53-responsive genes by
assistance. The Becton Dickinson FACS Vantage Cell Sorter was purchased genotoxic stress in hematopoietic cells containing wild-type and mutant p53. Gene
through a generous gift from the Keck Foundation. We thank Drs. C. Bevins, Expr., 8: 197–206, 1999.
Q. Chen, R. M. Macklis, S. Mazumder, G. Chen, and F. Dong for helpful
suggestions on the manuscript. 18. Chen, Q., Gong, B., and Almasan, A. Distinct stages of cytochrome c release from
mitochondria: evidence for a feedback amplification loop linking caspase activation
Note Added in Proof to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death
Differ., 7: 227–233, 2000.
After this work was completed, it was reported that radiation can sensitize breast
19. Chen, Q., Takeyama, N., Brady, G., Watson, A. J. M., and Dive, C. Blood cells with
carcinoma cells both in vitro and in vivo to Apo2L-induced apoptosis. This synergistic reduced mitochondrial membrane potential and cytosolic cytochrome c can survive
and maintain clonogenicity given appropriate signals to suppress apoptosis. Blood,
effect was p53-dependent and was suggested to be the result of radiation-induced up- 92: 4545– 4553, 1998.

regulation of DR5 (37). However, Apo2L is not induced following irradiation of these 20. Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. Bid, a Bcl2 interacting
breast carcinoma cells,5 indicating that the synergy between radiation and Apo2L might protein, mediates cytochrome c release from mitochondria in response to activation of
cell surface death receptors. Cell, 94: 481– 490, 1998.
work through different mechanisms in different cell types.
21. Owen-Schaub, L. B., Zhang, W., Cusack, J. C., Angelo, L. S., Santee, S. M.,
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Research.

Apo2 Ligand/TNF-related Apoptosis-inducing Ligand and
Death Receptor 5 Mediate the Apoptotic Signaling Induced by
Ionizing Radiation in Leukemic Cells

Bendi Gong and Alex Almasan
Cancer Res 2000;60:5754-5760.

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